Somatic hypermutation of variable genes, which encode a portion of immunoglobulin molecules, occurs at a frequency that is a million times greater than mutation in other genes. The molecular mechanism that introduces these mutations is unknown. Evidence points to a process that involves DNA repair events at sites of targeted strand breaks. In vertebrate cells, there are many recently identified DNA polymerases that inaccurately copy templates. One or more of these are potential candidates for enzymes that introduce base changes during hypermutation. We are studying the roles of DNA polymerases zeta, eta, and iota in the mechanism. (a) Polymerase zeta. Mice deficient for this enzyme die during mid-gestation, suggesting that the enzyme is critical for embryonic development. Interfering RNA to the sequence will be introduced into a mutating cell line to see the frequency of mutation is lowered. (b) Polymerase eta. This polymerase is defective in people with xeroderma pigmentosum variant disease. We sequenced variable genes from three patients and found that their frequency of hypermutation was normal, but the types of base changes were different. Polymerase eta-deficient clones had a decrease in the proportion of mutations at A and T with a concomitant rise of mutations at G and C. This finding implies that polymerase eta is an A-T mutator in hypermutation. We are using the yeast two-hybrid system to test for proteins that interact with the polymerase, and to see if it is involved in generating mutations in the heavy chain class switch region. (c) Polymerase iota. In collaboration with R. Woodgate, we have studied the specificity of polymerase iota on DNA substrates that might be formed during hypermutation. The fidelities of the polymerase are 10-fold lower when it fills a template at a DNA terminus compared to when it fills a longer template. We are currently studying the pattern of mutations in mice deficient for the polymerase.